The effectivity of thidiazuron and 1‐naphthaleneacetic acid on somatic embryo induction in transgenic Dendrobium phalaenopsis Fitzg. carrying 35S::GR::AtRKD4

Dendrobium phalaenopsis Fitzg. (also known as the Larat orchid) is an endemic orchid from Larat Island, Eastern Indonesia. Its beautiful flowers mean that many plants are taken for commercial purposes, leading to the rapid decline of populations in their natural habitats. The objectives of this study were to determine which organs of the transgenic Larat orchid carrying the 35S::GR::AtRKD4 construct, together with which concentrations of the plant growth regulators (PGRs) auxin and cytokinin, are suitable for the induction of somatic embryos (SEs). In this study, the AtRKD4 gene in Larat orchids was confirmed using PCR with specific primers for the AtRKD4 and HPT genes. Thidiazuron (TDZ) (1, 3 and 5 mg/L) in combination with 1‐naphthaleneacetic acid (NAA) (0.5 and 1 mg/L) were used on new phalaenopsis (NP) medium to induce SEs from leaves, pseudobulbs and roots. The AtRKD4 transgenes were detected as being stably integrated into the DNA genome of transformant plants using specific primers for AtRKD4 and HPT genes, and positive results were obtained using actin gene primers as internal controls for PCR. Pseudobulbs produced 19 to 20 SEs from 108 pseudobulb explants (89–100%), a higher number than produced in explants of the other organs studied. Among the PGR treatments, the best results were obtained in NP medium supplemented with a combination of 1 mg/L TDZ and 1 mg/L NAA, 100% of the explants of which produced SEs (2.11 ± 1.36). No significant difference was found between the morphology of the SEs produced from the non‐transformant Larat orchid pseudobulb explants and the 35S::AtRKD4 carrier transformant

Protoplasts Fusion of phalaenopsis and dendrobium = Fusi protoplas phalaenopsis dan dendrodium

Protoplasts of Phalaenopsis and Dendrobium were isolated enzymatically. The protoplasts of Phalaenopsis are easily distinguished from the protoplasts of Dendrobium because they were purple in colour, and after enzymatic digestion the chloroplast were in cluster at a certain site of the protoplast. The fusion of both protoplast were done using various concentration of Polyethylene Glycol (PEG) and osmoliticum stabilizer. The first fusion occured 5 minutes after both protoplasts were mixed in 35 % of PEG with 0,3 M glucose and 50 mM CaCI2 while other than 35 % were not effective. key words : protoplasts isolation - protoplasts fusio

In Vitro Culture of Phalaenopsis amabilis (L.) Blume Orchid for Seedling Production with Banana Extract Supplementation and Light Treatment for Ex Situ Conservation

In vitro culture is one of the effective cultivation methods for seedling production of orchids that can be used as a powerful tool in the conversation of orchids. The aims of the present study were to (i) investigate the effects of the addition of banana extract (0 g/l, 100 g/l, and 150 g/l) on media (NP media), in two different light regimes (light and dark conditions) on the growth of plantlets of an epiphytic orchid Phalaenopsis amabilis in in vitro culture. Methods used included (i) subculturing orchid seedlings in treatments media, (ii) measuring leaves and roots chlorophyll content and growth parameters, (iii) anatomical preparation of leaves and roots of the seedlings. The results showed that the best condition for getting greater seedlings of P. amabilis plantlets is in media with an addition of 100 g/L banana extract in light condition. The highest amount of chlorophyll in the P. amabilis leaves was found in medium with the addition of 100 g/L banana extract medium in light conditions. The thickness of mesophyll and the largest root diameter of P. amabilis seedlings were also found in media with the addition of 100 g/L banana extract medium in light condition. In conclusion, the addition of 100gr/L banana extract into basic culture medium will be beneficial for seedlings production of P. amabilis with great appearance, for ex situ orchid conservation programs

Induction of Somatic Embryogenesis through Overexpression of ATRKD4 Genes in Phalaenopsis “Sogo Vivien”

Phalaenopsis “Sogo Vivien “is a mini orchid hybrid with beautiful flowers and numerous inflorescences.Mass propagation of this orchid is needed to meet the market demand. Objective of this research was toinduce somatic embryogenesis of P.”Sogo Vivien” through insertion of AtRKD4 gene into orchid. T-DNAcontaining 35S::GAL4::AtRKD4::GR was inserted into 16-22 days after sowing orchid protocorms mediated byAgrobacterium tumefaciens EHA 105. Activation of the AtRKD4 gene was induced by glucocorticoid inductionsystem, using 15μM Dexamethasone (Dex). The results showed that 34 out of 2,648 orchid embryos developedinto protocorms on hygromycin selection medium, whereas only 4 out of 2,897 non-transformant protocormsdeveloped from embryos. A 500 bp of HPT genes was amplified from transformant candidates using specificprimers for HPT (HygF1 and HygR1) and 380 bp was amplified using specific primers for AtRKD4 (AtRKD4F1 and AtRKD4 R1), indicated that transgenes have been integrated into orchid genomes. Finally, 17 plantletswere positively carrying AtRKD4 and HPT genes, the efficiency of transformation was 0.63 %. Somatic embryoswere also emerged from leaf explants of transformant on hormone-free NP medium and became normalplantlets. It is probably due to the high activity of AtRKD4 genes in orchid

Micropropagation of Mini Orchid Hybrid Phalaenopsis “Sogo Vivien”

Phalaenopsis “Sogo Vivien” is an orchid hybrid with mini size plant body, and exhibits numerous beautiful pink flowers, that is ideal as ornamental pot plant. Some plants of this orchid exhibit variegated leaves that improve the beauty of the plant, not only because of the flower but also as attracted leaves. This orchid has high economical value, but mass propagation of this orchid has not established yet. An effective method to propagate both the normal and variegated plants is worth to be generated. The objective of this research was to produce a large number of P. “Sogo Vivien” plants, including the variegated plants. The method used seeds from self pollinating variegated plant, and flower stalk nodes. The seeds were sown on three various medium: VW, NP and MS, and flower stalk nodes were planted on VW + BA 10 mg l-1 + active carbon. The results showed that the best medium for in vitro culture of P. “Sogo Vivien” was NP medium, in which all seeds could grew into plantlets. Most plantlets emerged from the seeds were non variegated, only one plantlet out of 1344 seeds was variegated (0.007%). Although all emerged plantlets from flower stalk exhibited variegated leaves. Particularly, the plantlets arised from the second and third basal nodes of flower stalk showed the highest growth rate than that from the other nodes. Histological analysis showed that at 11-13 days after shoot segment plantation on NP medium, the shape of apical cells in the nodes was changed, then followed by the change of cell shape in the basal part of the nodes, produced bipolar pattern, then gradually developed into shoot. These results suggest that mass propagation could be achieved using seed culture, but to get the variegated phenotypes, the second and third nodes of flower stalk from variegated plant were the best explants to be used

Peptone and tomato extract induced early stage of embryo development of Dendrobium phalaenopsis Orchid

Germination and growth of orchid seeds can be accelerated by the addition of organic supplement and plant extract in culture medium. The objective of this study was to determine the effect of peptone and tomato extract on early stage of embryo development of Dendrobium phalaenopsis orchids. Orchid seeds were sown on NP and VW medium with addition of 10% of CW (NPCW and VWCW).  Five weeks after seed germination, about 58.03% seed germination was observed on VWCW medium, and only 37.45% seed germination on NPCW. Tomato extract and peptone were added in VWCW, resulting VWCWTP medium. After 4-8 weeks on VWCWTP, 94.42% seeds was germinated into plantlet, but only 67.30% germinated seeds on VWCW. To get optimal growth and development of  D.  phalaenopsis orchids embryos in the in vitro condition, supplement of 100 ml.L-1 coconut water, 100 mg.L-1 tomato extract and 2 mg.L-1 peptone into VW basic medium is required

Important Role of Mycorrhiza for Seed Germination and Growth of Dendrobium Orchids

Indonesia is a tropical country that has natural forests and is suitable for orchid species habitat, leading to more than 5,000 species of orchids grow. The tropical area is the main distribution centre for epiphytic orchids, one of which is Dendrobium, which grows more than 1,000 species throughout the world. Orchid seeds are very small and do not have an endosperm, making germination difficult in their natural habitat. Mycorrhizal association with orchids plays a role in the survival of orchids in nature through seed germination and growth. This study aims to provide a deeper understanding about the important role of mycorrhiza in seed germination and growth of Dendrobium. The mechanism of mycorrhizal association with orchids begins with the initial contact of the fungus with the orchid, hyphae enter the cortex cells to form peloton, peloton lysis, and exchange of nutrients occurs. Orchid mycorrhiza that mostly found groups in Dendrobium are Rhizoctonia (Epulorhiza, Tulasnella, Rhizoctonia). Mycorrhiza plays a role in increased secretion of phytohormone and enzyme activity which supports seed germination and growth of orchids. Specific mycorrhizal data on orchids can be used as an effort for in-situ and ex-situ conservation of Indonesian orchids, including Dendrobium

Induction of Microspore Embryogenesis of Eggplant (Solanum melongena L.) ‘Gelatik’

The haploid or double haploid plant of eggplants could be produced from microspore culture (embryogenesis of microspores). In the breeding programs, microspore can be developed into an embryo directly after exposure to stress treatment during cultured. Stress (temperature and starvation medium) is an important factor in the induction of embryogenesis microspore. This study aims to induced embryogenic microspores from eggplant CV. Gelatik. The stage late-uninucleate microspore (Vacuolate Microspore/VM) and early binucleate (Young Bicellular Pollen/YBP) are the suitable stages to induce multinucleate structure. There are 3 methods used in this research; 1) Determination of the stage development of microspore based on flower buds length and anther length. 2) Induction of embryogenic microspore on the pre-treatment and starvation medium. 3) After giving pre-treatment for 4 days, micropores were transferred to culture medium A2 at 28oC in dark conditions to induce the multicellular structures. This study reported that 50-68.51% of the VM+YBP stage obtained in the range of flower bud lengths of 10-17 mm, and 5.0-6.9 mm, the range of anther length containing VM+YBP of 50-77.48%. The pre-treatment heat shock at 33oC in the medium B for 2 days,  produced embryogenic microspores with a high percentage, that is about 50.19%, while microspores at 25oC and 4oC respectively 46.17% and 49.28%. Pre-treatment for 4 days at 4 oC, 25 oC,  and 33oC with the percentage of embryogenic microspores apiece 32.87%, 27.45%, and 37.34%. The multicellular (starlike) structure begins forming on the fifth day of incubation in culture medium (A2) after pre-treatment in B medium at 33oC

PENGARUH THIDIAZURON DAN NAPHTALENE ACETIC ACID UNTUK INDUKSI EMBRIOGENESIS SOMATIK DARI DAUN ANGGREK Phalaenopsis “Sogo Vivien”

Phalaenopsis “Sogo Vivien” merupakan salah satu anggrek hibrida hasil persilangan Phalaenopsis “Sogo Alice” X Phalaenopsis “Zuma‟s Pixie” dengan karakteristik morfologi berukuran mini serta memiliki bunga serta percabangan infloresensia yang banyak, sehingga dapat digunakan sebagai tanaman hias dalam pot. Embriogenesis somatik merupakan teknik mikropropagasi in vitro yang dapat menghasilkan anakan yang seragam dalam jumlah besar. Induksi embriogenesis somatik pada anggrek P. “Sogo Vivien” dapat dilakukan melalui perlakuan zat pengatur tumbuh Thidiazuron (TDZ) (0,5 dan 10 mg/l) yang dikombinasikan dengan Naphtalene Acetic Acid (NAA) 1 mg/l. Eksplan yang digunakan berupa daun dari seedling in vitro anggrek P. “Sogo Vivien”. Hasil penelitian menunjukkan bahwa TDZ 10 mg/l yang dikombinasikan dengan NAA 1 mg/l mampu menginduksi pembentukan embrio somatik secara langsung pada eksplan daun anggrek P. “Sogo Vivien” in vitro. Efisiensi pembentukan embrio somatik yang diperoleh adalah 5,7% dengan jumlah embrio somatik yang terbentuk adalah 57 embrio dari 1 eksplan daun. Hasil penelitian ini menunjukkan bahwa embrio somatik dapat diinduksi pada eksplan daun anggrek P. “Sogo Vivien”dengan menggunakan TDZ dan NAA Kata kunci : anggrek, embriogenesis somatik, thidiazuron, naphthalene-acetic-aci

Isolation and Characterization of Chalcone Synthase (CHS) Gene in Variegated-Flower of Dendrobium 'Enobi' and Phalaenopsis Hybrid Orchids

Variegated flowers, characterized by the presence of different colors in flowers, have high economic and aesthetic values. The main pigment in the orchid's purple flowers is anthocyanin, while the chalcone synthase (CHS) gene is the key to anthocyanin biosynthesis. Analysis of the CHS gene can reveal some changes, including mutations, in the process of color patterning in flowers. This study aims to determine the structure of the CHS gene related to color patterning in Dendrobium 'Enobi' and Phalaenopsis hybrid with variegated flowers. The methods applied in this study are floral morphology observation, DNA isolation, CHS gene amplification, anthocyanin measurement, and bioinformatic analysis. Morphologically, the variegated pattern has appeared since the flowers were still in the bud on both orchids. Based on the anthocyanin content analysis, the difference in the genus is not directly related to the differences in the flower's anthocyanin content. In addition, the purple zone in the D. 'Enobi' and Phalaenopsis hybrid has a longer fragment of CHS than the white zone. Our analysis suggested several mutations in the white zone and differences in the type and location of several conserved domain proteins. Mutations at the CHS gene fragment might cause decreased anthocyanin pigment formation in the white region